Mass spectrometric analysis of analytes such as small molecules as well as large biomolecules like proteins and oligonucleotides often involves sample preparation. It is axiomatic that the cleaner a sample is prior to analysis, then often the better analytical result is obtained.
Currently, sample purification of samples destined for mass spectrometric analysis in low volumes, i.e., <100 μL, prior to introducing the sample into a mass spectrometer, using an ionization technique such as MALDI or static nanospray, is often accomplished employing ZipTips® pipette columns from the Millipore Corporation. Alternatively, microcolumns prepared from gel-loader tips or short sections of fused silica packed with a chromatographic stationary phase are used to clean the sample. For static nanospray, the goal is to introduce the sample by electrospray ionization at low flow rates (20-40 nanoliters/minute) from a small initial volume of sample (˜1-5 μL) allowing extended MS analysis time. This is accomplished by placing the sample into a nanospray emitter which can be a pulled borosilicate glass tip coated with a conductive coating or from a “nozzle” in an array of microfabricated nozzles. ZipTips columns or other prepared microcolumns are currently used for removal of contaminants prior to mass spectrometric analysis.
One problem experienced by practitioners in the field in the application of ZipTips columns or microcolumns to the preparation of samples is poor recovery, particularly in the presence of buffers, detergents, salts, and chaotropes such as Tris, sodium dodecyl sulfate (SDS), urea, etc. For example, peptides extracted from one-dimensional or two-dimensional gels following in-gel digestion of separated proteins, wherein the gels contain around 0.1 percent of SDS, tend to have poor recovery using either ZipTips columns or microcolumns. Additionally, aspiration of samples through the packed bed of the ZipTips column often results in sample loss.
Moreover, the application of ZipTips pipette columns in concentrating a sample (>1.5 μL) is severely limited. The limitations articulated for ZipTips columns equally apply to microcolumn preparations. In order to effectuate the concentration of a sample using either ZipTips columns or a microcolumn, multiple steps of passing a sample through one of these devices must occur. As this reiteration occurs, progressively more sample can be lost resulting in poor recovery. Further, aspirating a sample through a ZipTips columns or microcolumn often results in air being introduced into the packing bed resulting in poor retention/recovery of the analytes of interest.
A further issue encountered by practitioners in this field is difficulty in manipulating microliter volumes through ZipTips columns or a microcolumn with the concomitant loss of sample.
Currently there is a need for a sample preparation device that effectively removes contaminants from the sample, concentrates the sample and permits easy manipulation of small volumes of liquid. The present invention addresses the aforementioned issues and provides a solution that effectively eliminates concerns present in the prior art.